Investigation of HIV-TB co-infection through analysis of the potential impact of host genetic variation on host-pathogen protein interactions

HIV and Mycobacterium tuberculosis (Mtb) co-infection causes treatment and diagnostic difficulties, which places a major burden on health care systems in settings with high prevalence of both infectious diseases, such as South Africa. Human genetic variation adds further complexity, with variants affecting disease susceptibility and response to treatment. The identification of variants in African populations is affected by reference mapping bias, especially in complex regions like the Major Histocompatibility Complex (MHC), which plays an important role in the immune response to HIV and Mtb infection. We used a graph-based approach to identify novel variants in the MHC region within African samples without mapping to the canonical reference genome. We generated a host-pathogen functional interaction network made up of inter- and intraspecies protein interactions, gene expression during co-infection, drug-target interactions, and human genetic variation. Differential expression and network centrality properties were used to prioritise proteins that may be important in co-infection. Using the interaction network we identified 28 human proteins that interact with both pathogens (”bridge” proteins). Network analysis showed that while MHC proteins did not have significantly higher centrality measures than non-MHC proteins, bridge proteins had significantly shorter distance to MHC proteins. Proteins that were significantly differentially expressed during co-infection or contained variants clinically-associated with HIV or TB also had significantly stronger network properties. Finally, we identified common and consequential variants within prioritised proteins that may be clinically-associated with HIV and TB. The integrated network was extensively annotated and stored in a graph database that enables rapid and high throughput prioritisation of sets of genes or variants, facilitates detailed investigations and allows network-based visualisation

Analysis of High-Throughput Data - Protein-Protein Interactions, Protein Complexes and RNA Half-life

The development of high-throughput techniques has lead to a paradigm change in biology from the small-scale analysis of individual genes and proteins to a genome-scale analysis of biological systems. Proteins and genes can now be studied in their interaction with each other and the cooperation within multi-subunit protein complexes can be investigated. Moreover, time-dependent dynamics and regulation of these processes and associations can now be explored by monitoring mRNA changes and turnover. The in-depth analysis of these large and complex data sets would not be possible without sophisticated algorithms for integrating different data sources, identifying interesting patterns in the data and addressing the high variability and error rates in biological measurements. In this thesis, we developed such methods for the investigation of protein interactions and complexes and the corresponding regulatory processes. In the first part, we analyze networks of physical protein-protein interactions measured in large-scale experiments. We show that the topology of the complete interactomes can be confidently extrapolated despite high numbers of missing and wrong interactions from only partial measurements of interaction networks. Furthermore, we find that the structure and stability of protein interaction networks is not only influenced by the degree distribution of the network but also considerably by the suppression or propagation of interactions between highly connected proteins. As analysis of network topology is generally focused on large eukaryotic networks, we developed new methods to analyze smaller networks of intraviral and virus-host interactions. By comparing interactomes of related herpesviral species, we could detect a conserved core of protein interactions and could address the low coverage of the yeast two-hybrid system. In addition, common strategies in the interaction of the viruses with the host cell were identified. New affinity purification methods now make it possible to directly study associations of proteins in complexes. Due to experimental errors the individual protein complexes have to be predicted with computational methods from these purification results. As previously published methods relied more or less heavily on existing knowledge on complexes, we developed an unsupervised prediction algorithm which is independent from such additional data. Using this approach, high-quality protein complexes can be identified from the raw purification data alone for any species purification experiments are performed. To identify the direct, physical interactions within these predicted complexes and their subcomponent structure, we describe a new approach to extract the highest scoring subnetwork connecting the complex and interactions not explained by alternative paths of indirect interactions. In this way, important interactions within the complexes can be identified and their substructure can be resolved in a straightforward way. To explore the regulation of proteins and complexes, we analyzed microarray measurements of mRNA abundance, de novo transcription and decay. Based on the relationship between newly transcribed, pre-existing and total RNA, transcript half-life can be estimated for individual genes using a new microarray normalization method and a quality control can be applied. We show that precise measurements of RNA half-life can be obtained from de novo transcription which are of superior accuracy to previously published results from RNA decay. Using such precise measurements, we studied RNA half-lives in human B-cells and mouse fibroblasts to identify conserved patterns governing RNA turnover. Our results show that transcript half-lives are strongly conserved and specifically correlated to gene function. Although transcript half-life is highly similar in protein complexes and \mbox{families}, individual proteins may deviate significantly from the remaining complex subunits or family members to efficiently support the regulation of protein complexes or to create non-redundant roles of functionally similar proteins. These results illustrate several of the many ways in which high-throughput measurements lead to a better understanding of biological systems. By studying large-scale measure\-ments in this thesis, the structure of protein interaction networks and protein complexes could be better characterized, important interactions and conserved strategies for herpes\-viral infection could be identified and interesting insights could be gained into the regulation of important biological processes and protein complexes. This was made possible by the development of novel algorithms and analysis approaches which will also be valuable for further research on these topics